In vitro phosphorylation of human complement factor C3 by protein kinase A and protein kinase C. Effects on the classical and alternative pathways.
Complement factor C3, recently found to contain covalently bound phosphate, was phosphorylated in vitro by cyclic AMP-dependent protein kinase (protein kinase A) and Ca2(+)-activated, phospholipid-dependent protein kinase (protein kinase C). Both protein kinases phosphorylated the same serine residue(s) located in the C3a portion of the alpha-chain. In addition, protein kinase C phosphorylated the beta-chain to a lesser extent. Protein kinase A gave a maximal incorporation of 1 mol of phosphate/mol of C3 while that value with protein kinase C was 1.5 mol of phosphate/mol of C3. The velocity in pmol of [32P]phosphate/(min x unit kinase) was 20 times higher for protein kinase C than for protein kinase A although a 10 times lower ratio of protein kinase to C3 was used in the former case. The apparent Km for C3 was 2.6 microM when protein kinase C was used. The phosphorylated C3 was found to be more resistant to partial degradation by trypsin than unphosphorylated C3. It was also found that phosphorylation of C3 in the C3a portion of the alpha-chain inhibited both the classical and alternative complement activation pathways on an approximately stoichiometric basis.
Forsberg
P O
PO
Department of Medical and Physiological Chemistry, Uppsala, Sweden.
Martin
S C
SC
Nilsson
B
B
Ekman
P
P
Nilsson
U R
UR
Engström
L
L
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D003167:Complement Activation; D003176:Complement C3 / Q000378:metabolism*; D003170:Complement Pathway, Alternative; D003171:Complement Pathway, Classical; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009132:Muscles / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D013154:Spleen / Q000201:enzymology; D013552:Swine; D014357:Trypsin
D000596:Amino Acids; D003176:Complement C3; D010446:Peptide Fragments; D011494:Protein Kinases; D011493:Protein Kinase C; D014357:Trypsin